Open Resource CRISPR/CAS9 Genome-Wide sgRNA Library Screening Platform. ABSTRACT CRISPR technology made its giant leap from bacteria to mammalian system when a few landmark discoveries that demonstrated Cas9 protein from Streptococcus Pyogenes could be reprogrammed with synthetic RNAs (sgRNAs) to generate site-specific double-strand breaks (DSBs) in vitro and in mammalian cells. CRISPR technology appealed to the world of genomics and gene editing as it made the task of generating large genome-editing libraries possible for functional genetic screens. Although there has been a tremendous increase in CRISPR related publications over the last two years in many areas of drug discovery and gene-function analysis, it is already being recognized by the scientific community that the application of the technology to high throughput functional genomics screening comes with its own unique challenges and a significant improvement is warranted. As research in this field focuses on improving effectiveness, increasing selectivity and reducing off- target effects, companies aim to develop simplified, flexible, robust and cost-effective CRISPR screening platforms, incorporating all of the above with experimental and bioinformatics tools for data validation and integration of this information into operational cell-based models. CRISPR technology can be used to either achieve sgRNA directed gene knockout by Cas9 endonuclease (CRISPR-KO) or sgRNA directed regulation of gene expression by mutant dCas9 (CRISPRa for transactivation, CRISPRi for inhibition). While published studies have provided proof that both CRISPR-KO and CRISPRa/i can be successfully used in high-throughput functional genomics screens, substantial margins of improvements do exist in multiple aspects of the technology. Additionally, although the CRISPR system has opened many potential avenues for improving the drug discovery process, those remain only potential opportunities until we develop robust commercially available CRISPR screening technologies, as well as experimental and bioinformatics tools for data validation and integration of this information into operational cell-based models. Following the successful completion of Phase I studies, in the present Phase II application we propose the final development and commercialization of a high performance CRISPR screening platform with innovative features enhancing the frequency of effective gene knockout in CRISPR-KO applications, and the intensity of promoter activation/repression in CRSPRa/i. As supporting tools, we will provide protocols, reagents and software tools for screening data analysis and validation. The ultimate goal of this project is to develop, validate and commercialize a set of high efficiency, ready- to-screen, genome-wide pooled sgRNA human and mouse CRISPR lentiviral libraries, supporting bioinformatics tools for data analysis, as well as custom services for CRISPR library design, CRISPR functional genetic screening, and hit confirmation/validation assays. Upon completion of the proposed Phase II studies, Cellecta will be ready to provide the scientific community with a comprehensive, highly efficient, scalable and cost- effective genetic screening tool set that is expected to expedite systematic identification of new targets for therapeutic intervention and facilitate the development of highly specific drugs, biomarkers and novel therapeutic concepts. The new CRISPR screening platform, including sgRNA libraries, custom services, supporting software and data set tools developed in the course of Phase I-II studies will be made available to all academic and commercial researchers through www.cellecta.com web site.